PCR from genomic template:
Initially, try a regular PCR protocol:
1 ul genomic DNA
1.5 ul 10uM each primer
10 ul Phusion buffer
5 ul 2mM dNTPs
dH2O to 49ul
add 1 ul Phusion
PCR program:
94o 30 sec
94o 30 sec
5o degrees below the melting temperature of your primers 30 sec
72o 1 min (the standard is 30 sec-1 min/ kb of PCR product)
35 cycles
72o 10 min
Hold at 15o
If that doesn’t work, try Touchdown PCR:
94 o 30 sec
94o 30 sec
1o degree above the melting temperature of your primers
and subtract 0.5o with every cycle (total of 5o)
30 sec
o
72 1 min (the standard is 30 sec – 1 min/ kb of PCR product)
10 cycles
98o 30 sec
4o degrees below the melting temperature of your primers 30 sec
72o 1 min (the standard is 30 sec – 1 min/ kb of PCR product)
25 cycles
72o 10 min
Hold at 15o